Methods for reducing remnant cholesterol and other lipoprotein fractions by administering an inhibitor of proprotein convertase subtilisin kexin-9 (PCSK9)

ABSTRACT

The present invention provides methods for reducing various lipoprotein fractions in the serum of patients. The methods of the invention include reducing serum remnant cholesterol, and/or the serum concentration of one or more LDL-C subfractions in a patient. The methods of the present invention comprise selecting a patient who exhibits elevated serum lipoproteins, and administering to the patient a pharmaceutical composition comprising a PCSK9 inhibitor. In certain embodiments, the PCSK9 inhibitor is an anti-PCSK9 antibody such as the exemplary antibody referred to herein as mAb316P.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.provisional application Nos. 61/828,753, filed on May 30, 2013;61/901,705, filed on Nov. 8, 2013; 61/919,836, filed on Dec. 23, 2013;61/935,358, filed on Feb. 4, 2014; 61/953,959, filed on Mar. 17, 2014;and 61/991,738, filed on May 12, 2014, the disclosures of which areherein incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to the field of therapeutic treatments ofdiseases and disorders which are associated with elevated levels oflipoproteins. More specifically, the invention relates to theadministration of PCSK9 inhibitors to reduce the levels of serum remnantcholesterol and other lipoprotein subfractions in a patient.

BACKGROUND

Remnant cholesterol (also referred to as remnant lipoprotein) is acategory of cholesterol that comprises non-HDL and non-LDL cholesterol.Remnant lipoproteins are products of VLDL lipolysis, and include VLDL₃and intermediate-density lipoproteins (IDL, the direct precursor to LDLformation). Serum remnant cholesterol level has been identified as apredictive risk factor for coronary artery disease. In addition toremnant cholesterol, there are several subfractions of low densitylipoprotein cholesterol (LDL-C) that may have relevance with regard tocardiovascular health. In particular, LDL-C is composed of a continuumof LDL particles of different densities and states of lipidation.Therapeutic reduction of serum remnant cholesterol and other lipoproteinsubfraction levels may be a means for treating or reducing the risk ofcardiovascular disorders.

PCSK9 is a proprotein convertase belonging to the proteinase K subfamilyof the secretory subtilase family. The use of PCSK9 inhibitors(anti-PCSK9 antibodies) to reduce serum total cholesterol, LDLcholesterol and serum triglycerides is described in U.S. Pat. Nos.8,062,640, 8,357,371, and US Patent Application Publication No.2013/0064834.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods for reducing various lipoproteinsand lipoprotein fractions in the serum of a patient. The patient can bea patient with hypercholesterolemia or at risk of developinghypercholesterolemia, or who has or is at risk of developing acardiovascular disease.

The methods of the present invention comprise selecting a patient withan elevated serum level of a particular lipoprotein or lipoproteinfraction, and administering to the patient a pharmaceutical compositioncomprising an inhibitor of proprotein convertase subtilisin kexin-9(PCSK9). According to certain aspects of the invention, the patient isselected on the basis of having an elevated serum remnant cholesterollevel. According to certain other aspects of the invention, the patientis selected on the basis of having an elevated serum very low-densitylipoprotein cholesterol level (e.g., elevated serum VLDL-C, VLDL₁-C,VLDL₂-C, VLDL₁₊₂-C, VLDL₃-C, etc.). According to yet other aspects ofthe invention, the patient is selected on the basis of having anelevated serum intermediate-density lipoprotein cholesterol (IDL-C)level. According to certain other aspects of the invention, the patientis selected on the basis of having an elevated serum level of one ormore low-density lipoprotein cholesterol (LDL-C) subfraction (e.g.,elevated serum LDL₁-C, LDL₂-C, LDL₃-C, LDL₄-C, LDL₃₊₄-C, etc.).According to certain aspects of the present invention, the patient isadditionally or alternatively selected on the basis of having anelevated serum level of Lp(a).

The patient may also be selected on the basis of exhibiting additionalrisk factors for such diseases and disorders in which a reduction inlipoprotein levels would be beneficial or risk-lowering. For example,patients with hypercholesterolemia (e.g., heFH, nonFH, etc.) may be goodcandidates for treatment with the therapeutic methods of the presentinvention.

PCSK9 inhibitors which may be administered in accordance with themethods of the present invention include, e.g., anti-PCSK9 antibodies orantigen-binding fragments thereof. Specific exemplary anti-PCSK9antibodies which may be used in the practice of the methods of thepresent invention include any antibodies or antigen-binding fragments asset forth in U.S. Pat. No. 8,062,640, and/or disclosed herein.

The PCSK9 inhibitor may be administered to a subject subcutaneously orintravenously. Furthermore, the PCSK9 inhibitor may be administered to apatient who is on a therapeutic statin regimen at the time oftherapeutic intervention.

Other embodiments of the present invention will become apparent from areview of the ensuing detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the dynamic relationship between mAb316P, free (unbound)PCSK9, and LDL-C levels following a single dose of mAb316P 150 mg SC inhealth subjects who were not receiving background statin therapy.

FIG. 2 shows the mean concentration of free mAb316P in HeFH and non-FHpatients receiving mAb316P 50, 100, or 150 mg SC plus atorvastatin ormAb316P 150 mg SC plus diet only. Dotted line indicates quantitationlimit for free PCSK9 (0.0312 mg/dL)

FIG. 3 shows the mean percentage change in LDL-C from baseline versusmean total mAb316P concentration (mg/dL) in patients (HeFH and non-FHcombined; n−21 receiving mAb316P 150 mg SC on Study Days 1, 29, and 43.Points indicate sampling times; h, hour; d, day. The hysteresis curvemoves in a clockwise direction over time, as indicated by the arrows,demonstrating the temporal relationship between mAb316P concentrationand change in LDL-C.

FIG. 4 shows mean baseline free PCSK9 levels in non-FH patientsreceiving atorvastatin or diet alone (no statin).

FIG. 5 shows LDL-C efficacy curves for patients receiving mAb316P 150 mgSC or placebo±atorvastatin.

FIG. 6 shows the mean percentage change in LDL-C from baseline over timein hypercholesterolemic patients on concomitant statin therapy followingadministration of mAb316P 50 mg, 100 mg, or 150 mg Q2W, 200 mg or 300 mgQ4W, or placebo. Delta symbol (Δ) corresponds to LOCF means.

FIGS. 7-9 show the mean (SD) levels of cholesterol in VLDL and serumremnant lipoprotein subfractions and triglycerides before and aftertreatment for placebo and mAb316P 150 mg Q2W in three different clinicalstudies (A, B, and C, as summarized in Table 1 herein). FIG. 7 depictsthe results for Study A, FIG. 8 depicts the results for Study B, andFIG. 9 depicts the results for Study C.

FIGS. 10-12 show the mean (SD) levels of cholesterol in LDL subfractionsbefore and after treatment for placebo and mAb316P 150 mg Q2W in threedifferent clinical studies (A, B, and C, as summarized in Table 1herein). FIG. 10 depicts the results for Study A, FIG. 11 depicts theresults for Study B, and FIG. 12 depicts the results for Study C.

FIG. 13, panels A and B, show the dose response changes in apo CII andapo CIII, respectively, for all doses in Study A (as summarized in Table1 herein).

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice of the present invention,the preferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to describe intheir entirety.

Methods for Reducing Remnant Cholesterol and Other Lipoprotein Fractions

The present invention provides methods for reducing serum remnantcholesterol and other lipoprotein fractions in a patient. The methods ofthe invention comprise selecting a patient who exhibits an elevatedserum level of one or more of remnant cholesterol (also referred to as“remnant lipoprotein cholesterol,” “remnant lipoproteins,” or RLP-C),very low density lipoprotein cholesterol (VLDL-C), intermediate densitylipoprotein cholesterol (IDL-C), triglycerides (TG), and or Lp(a). Incertain embodiments, the methods of the invention comprise selecting apatient who exhibits elevated serum VLDL₁, VLDL₂, VLDL₁₊₂, VLDL₃, LDL₁,LDL₂, LDL₃, LDL₄, LDL₃₊₄, and/or other combinations thereof. The methodsof the present invention further comprise administering to the patient apharmaceutical composition comprising a PCSK9 inhibitor.

As used herein, the expressions “remnant cholesterol,” “remnantlipoproteins,” “RLP,” and the like, means the cholesterol content oftriglyceride-rich lipoproteins, composed of VLDLs and IDLs in thefasting state and of these two lipoproteins together with chylomicronremnants in the nonfasting state. Remnant cholesterol can be calculatedas: total cholesterol minus HDL-C minus LDL-C (i.e., non-[HDL-C+LDL-C]).Remnant cholesterol includes VLDL₃ and IDL.

Other lipoprotein fractions include, e.g., LDL₁-C (“large buoyant” LDL),LDL₂-C, LDL₃-C and LDL₄-C (“small dense” LDL). The serum levels of theselipoproteins may be ascertained by standard lipoprotein subfractionationtechniques such as, e.g., vertical auto profile (VAP) testing, ionmobility, etc.

In the context of the present invention, “elevated serum lipoprotein”(e.g., elevated serum remnant cholesterol, elevated serum VLDL-C,elevated serum VLDL₁-C, elevated serum VLDL₂-C, elevated serumVLDL₁₊₂-C, elevated serum VLDL₃-C, elevated serum IDL-C, elevated serumLDL₁-C, elevated serum LDL₂-C, elevated serum LDL₃-C, elevated serumLDL₄-C, elevated serum LDL₃₊₄-C, etc.) means a serum level of therespective lipoprotein greater than about 8 mg/dL. In certainembodiments, a patient is considered to exhibit an elevated serumlipoprotein if the level of the particular lipoprotein measured in theserum of a patient is greater than about 9 mg/dL, 10 mg/dL, 11 mg/dL, 12mg/dL, 13 mg/dL, 14 mg/dL, 15 mg/dL, 16 mg/dL, 17 mg/dL, 18 mg/dL, 19mg/dL, 20 mg/dL, 25 mg/dL, 30 mg/dL, 35 mg/dL, 40 mg/dL, 45 mg/dL, 50mg/dL. The serum lipoprotein level can be measured in a patientpost-prandial. In some embodiments, the lipoprotein level is measuredafter a period of time of fasting (e.g., after fasting for 6 hrs, 8 hrs,10 hrs, 12 hrs or more). Any clinically acceptable diagnostic method canbe used in the context of the present invention to measure serumlipoprotein in a patient.

According to the present invention, a reduction in serum remnantcholesterol or other lipoprotein subfraction (e.g., VLDL-C, VLDL₁-C,VLDL₂-C, VLDL₁₊₂-C, VLDL₃-C, IDL-C, IDL₁-C, IDL₂-C, IDL₁₊₂-C, LDL₁-C,LDL₂-C, LDL_(2a)-C, LDL_(2b)-C, LDL_(1+2a)-C, LDL₃-C, LDL_(3a)-C,LDL_(3b)-C, LDL₄-C, LDL_(4a)-C, LDL_(4b)-C, LDL_(4c)-C,LDL_(4a+4b+4c+3b)-C, LDL₃₊₄-C, etc.) means that the serumlipoprotein/lipoprotein subfraction level in the patient after receivinga pharmaceutical composition of the invention is reduced by about 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, or more, from baseline. As used herein, “baseline level,” whenreferring to a particular lipoprotein or lipoprotein subfraction, meansthe level of lipoprotein/lipoprotein subfraction measured in the serumof a subject prior to receiving a pharmaceutical composition of thepresent invention. The reduction in serum lipoprotein/lipoproteinsubfraction resulting from administration of a pharmaceuticalcomposition of the invention may be achieved and/or observed at 1 day, 2days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 4 weeks, 6 weeks,8 weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22weeks, or longer, following the initiation of a therapeutic regimencomprising administration of one or more doses of a pharmaceuticalcomposition comprising a PCSK9 inhibitor, examples of which aredisclosed elsewhere herein.

For example, the present invention includes methods for reducing serumremnant cholesterol in a patient by selecting a patient with elevatedserum remnant cholesterol, and administering to the patient apharmaceutical composition comprising a PCSK9 inhibitor, wherein serumremnant cholesterol is reduced in the patient by at least about 35% toabout 45% from baseline following administration of the pharmaceuticalcomposition.

The present invention also includes methods for reducing serum VLDL-C ina patient by selecting a patient with elevated serum VLDL-C, andadministering to the patient a pharmaceutical composition comprising aPCSK9 inhibitor, wherein serum VLDL-C is reduced in the patient by atleast about 20% to about 28% from baseline following administration ofthe pharmaceutical composition.

The present invention also includes methods for reducing serum VLDL₁₊₂-Cin a patient by selecting a patient with elevated serum VLDL₁₊₂-C, andadministering to the patient a pharmaceutical composition comprising aPCSK9 inhibitor, wherein serum VLDL₁₊₂-C is reduced in the patient by atleast about 20% to about 32% from baseline following administration ofthe pharmaceutical composition.

The present invention also includes methods for reducing serum VLDL₃-Cin a patient by selecting a patient with elevated serum VLDL₃-C, andadministering to the patient a pharmaceutical composition comprising aPCSK9 inhibitor, wherein serum VLDL₃-C is reduced in the patient by atleast about 20% to about 27% from baseline following administration ofthe pharmaceutical composition.

The present invention also includes methods for reducing serum IDL-C ina patient by selecting a patient with elevated serum IDL-C, andadministering to the patient a pharmaceutical composition comprising aPCSK9 inhibitor, wherein serum IDL-C is reduced in the patient by atleast about 50% to about 56% from baseline following administration ofthe pharmaceutical composition.

The present invention also includes methods for reducing serum LDL₁-C ina patient by selecting a patient with elevated serum LDL₁-C, andadministering to the patient a pharmaceutical composition comprising aPCSK9 inhibitor, wherein serum LDL₁-C is reduced in the patient by atleast about 65% to about 78% from baseline following administration ofthe pharmaceutical composition.

The present invention also includes methods for reducing serum LDL₂-C ina patient by selecting a patient with elevated serum LDL₂-C, andadministering to the patient a pharmaceutical composition comprising aPCSK9 inhibitor, wherein serum LDL₂-C is reduced in the patient by atleast about 75% to about 85% from baseline following administration ofthe pharmaceutical composition.

The present invention also includes methods for reducing serum LDL₃₊₄-Cin a patient by selecting a patient with elevated serum LDL₃₊₄-C, andadministering to the patient a pharmaceutical composition comprising aPCSK9 inhibitor, wherein serum LDL₃₊₄-C is reduced in the patient by atleast about 45% to about 70% from baseline following administration ofthe pharmaceutical composition.

The present invention also includes methods for reducing apolipoprotein(apo) CII and/or CIII in a patient by selecting a patient withhypercholesterolemia, and administering to the patient a pharmaceuticalcomposition comprising a PCSK9 inhibitor. According to certainembodiments, the patient has non-familial hypercholesterolemia (non-FH);in certain other embodiments, the patient has heterozygous familialhypercholesterolemia (he-FH). According to certain embodiments of thisaspect of the invention, apo CII is reduced in the patient by at leastabout 9% to about 30% from baseline, and apo CIII is reduced in thepatient by at least about 20% to about 25%, following administration ofthe pharmaceutical composition.

The present invention also includes methods for reducing serum Lp(a) ina patient by selecting a patient with elevated serum Lp(a), andadministering to the patient a pharmaceutical composition comprising aPCSK9 inhibitor, wherein serum Lp(a) is reduced in the patient by atleast about 10% to about 40% (e.g., about 25%, about 30% or about 35%)from baseline following administration of the pharmaceuticalcomposition.

The present invention also includes methods for reducing lipoproteinparticle concentration in a patient by selecting a patient withhypercholesterolemia and/or elevated serum lipoprotein particleconcentration, and administering to the patient a pharmaceuticalcomposition comprising a PCSK9 inhibitor. The methods according to thisaspect of the invention are useful for, inter alia, reducing theconcentration of low-density lipoprotein particles (LDL-P), intermediatedensity lipoprotein particles (IDL-P), and very low-density lipoproteinparticles (VLDL-P), including methods for reducing the serumconcentrations of, e.g., small VLDL-P (diameter 29 to 42 mm); mediumVLDL-P (diameter 42 to 60 nm); large VLDL-P (diameter>60 nM); smallLDL-P (diameter 18 to 20.5 nm); large LDL-P (diameter 20.5 to 23 nm);and IDL-P (diameter 23 to 29 nm).

Patient Population

The methods of the present invention are useful for reducing serumremnant cholesterol and other lipoprotein fractions (e.g., VLDL-C,VLDL₁-C, VLDL₂-C, VLDL₁₊₂-C, VLDL₃-C, IDL-C, LDL₁-C, LDL₂-C, LDL₃-C,LDL₄-C, LDL₃₊₄-C, etc.) in a patient. In some instances the patient isotherwise healthy except for exhibiting elevated levels of one or moreof the aforementioned serum lipoproteins. For example, the patient maynot exhibit any other risk factor of cardiovascular, thrombotic or otherdiseases or disorders at the time of treatment. In other instances,however, the patient is selected on the basis of being diagnosed with,or at risk of developing, a disease or disorder that is caused by orcorrelated with elevated serum remnant cholesterol or with elevatedserum levels of other lipoproteins or lipoprotein fractions. Forexample, at the time of, or prior to administration of thepharmaceutical composition of the present invention, the patient may bediagnosed with or identified as being at risk of developing acardiovascular disease or disorder, such as, e.g., coronary arterydisease, acute myocardial infarction, asymptomatic carotidatherosclerosis, stroke, peripheral artery occlusive disease, etc. Thecardiovascular disease or disorder, in some instances, ishypercholesterolemia. For example, a patient may be selected fortreatment with the methods of the present invention if the patient isdiagnosed with or identified as being at risk of developing ahypercholesterolemia condition such as, e.g., heterozygous FamilialHypercholesterolemia (heFH), homozygous Familial Hypercholesterolemia(hoFH), Autosomal Dominant Hypercholesterolemia (ADH, e.g., ADHassociated with one or more gain-of-function mutations in the PCSK9gene), as well as incidences of hypercholesterolemia that are distinctfrom Familial Hypercholesterolemia (nonFH).

In other instances, at the time of, or prior to administration of thepharmaceutical composition of the present invention, the patient may bediagnosed with or identified as being at risk of developing an arterialdisorder such as cerebrovascular occlusive disease, peripheral vasculardisease, peripheral arterial disorder, etc. In certain embodiments, thepatient is selected on the basis of being diagnosed with or at risk ofdeveloping a combination of two or more of the abovementioned diseasesor disorders.

In yet other instances, the patient who is to be treated with themethods of the present invention is selected on the basis of one or morefactors selected from the group consisting of age (e.g., older than 40,45, 50, 55, 60, 65, 70, 75, or 80 years), race, gender (male or female),exercise habits (e.g., regular exerciser, non-exerciser), otherpreexisting medical conditions (e.g., type-II diabetes, high bloodpressure, etc.), and current medication status (e.g., currently takingstatins [e.g., cerivastatin, atorvastatin, simvastatin, pitavastatin,rosuvastatin, fluvastatin, lovastatin, pravastatin, etc.], betablockers, niacin, etc.). The present invention also includes methods forreducing serum remnant cholesterol and/or other lipoprotein fractionlevels in patients who are intolerant of, non-responsive to, orinadequately responsive to conventional statin therapy. Potentialpatients can be selected/screened on the basis of one or more of thesefactors (e.g., by questionnaire, diagnostic evaluation, etc.) beforebeing treated with the methods of the present invention.

PCSK9 Inhibitors

The methods of the present invention comprise administering to a patienta therapeutic composition comprising a PCSK9 inhibitor. As used herein,a “PCSK9 inhibitor” is any agent which binds to or interacts with humanPCSK9 and inhibits the normal biological function of PCSK9 in vitro orin vivo. Non-limiting examples of categories of PCSK9 inhibitors includesmall molecule PCSK9 antagonists, peptide-based PCSK9 antagonists (e.g.,“peptibody” molecules), and antibodies or antigen-binding fragments ofantibodies that specifically bind human PCSK9.

The term “human proprotein convertase subtilisin/kexin type 9” or “humanPCSK9” or “hPCSK9”, as used herein, refers to PCSK9 having the nucleicacid sequence shown in SEQ ID NO:754 and the amino acid sequence of SEQID NO:755, or a biologically active fragment thereof.

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprising four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds,as well as multimers thereof (e.g., IgM). Each heavy chain comprises aheavy chain variable region (abbreviated herein as HCVR or V_(H)) and aheavy chain constant region. The heavy chain constant region comprisesthree domains, C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises alight chain variable region (abbreviated herein as LCVR or V_(L)) and alight chain constant region. The light chain constant region comprisesone domain (C_(L)1). The V_(H) and V_(L) regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDRs), interspersed with regions that are moreconserved, termed framework regions (FR). Each V_(H) and V_(L) iscomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. In different embodiments of the invention, the FRs of theanti-PCSK9 antibody (or antigen-binding portion thereof) may beidentical to the human germline sequences, or may be naturally orartificially modified. An amino acid consensus sequence may be definedbased on a side-by-side analysis of two or more CDRs.

The term “antibody,” as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDRwhich is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (v)V_(H)-C_(H)1-C_(H)2-C_(H)3; (vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L);(viii) V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemonospecific or multispecific (e.g., bispecific). A multispecificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multispecific antibody format, including theexemplary bispecific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques available in the art.

The constant region of an antibody is important in the ability of anantibody to fix complement and mediate cell-dependent cytotoxicity.Thus, the isotype of an antibody may be selected on the basis of whetherit is desirable for the antibody to mediate cytotoxicity.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay nonetheless include amino acid residues not encoded by humangermline immunoglobulin sequences (e.g., mutations introduced by randomor site-specific mutagenesis in vitro or by somatic mutation in vivo),for example in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell (describedfurther below), antibodies isolated from a recombinant, combinatorialhuman antibody library (described further below), antibodies isolatedfrom an animal (e.g., a mouse) that is transgenic for humanimmunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res.20:6287-6295) or antibodies prepared, expressed, created or isolated byany other means that involves splicing of human immunoglobulin genesequences to other DNA sequences. Such recombinant human antibodies havevariable and constant regions derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies are subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

Human antibodies can exist in two forms that are associated with hingeheterogeneity. In one form, an immunoglobulin molecule comprises astable four chain construct of approximately 150-160 kDa in which thedimers are held together by an interchain heavy chain disulfide bond. Ina second form, the dimers are not linked via inter-chain disulfide bondsand a molecule of about 75-80 kDa is formed composed of a covalentlycoupled light and heavy chain (half-antibody). These forms have beenextremely difficult to separate, even after affinity purification.

The frequency of appearance of the second form in various intact IgGisotypes is due to, but not limited to, structural differencesassociated with the hinge region isotype of the antibody. A single aminoacid substitution in the hinge region of the human IgG4 hinge cansignificantly reduce the appearance of the second form (Angal et al.(1993) Molecular Immunology 30:105) to levels typically observed using ahuman IgG1 hinge. The instant invention encompasses antibodies havingone or more mutations in the hinge, C_(H)2 or C_(H)3 region which may bedesirable, for example, in production, to improve the yield of thedesired antibody form.

An “isolated antibody,” as used herein, means an antibody that has beenidentified and separated and/or recovered from at least one component ofits natural environment. For example, an antibody that has beenseparated or removed from at least one component of an organism, or froma tissue or cell in which the antibody naturally exists or is naturallyproduced, is an “isolated antibody” for purposes of the presentinvention. An isolated antibody also includes an antibody in situ withina recombinant cell. Isolated antibodies are antibodies that have beensubjected to at least one purification or isolation step. According tocertain embodiments, an isolated antibody may be substantially free ofother cellular material and/or chemicals.

The term “specifically binds,” or the like, means that an antibody orantigen-binding fragment thereof forms a complex with an antigen that isrelatively stable under physiologic conditions. Methods for determiningwhether an antibody specifically binds to an antigen are well known inthe art and include, for example, equilibrium dialysis, surface plasmonresonance, and the like. For example, an antibody that “specificallybinds” PCSK9, as used in the context of the present invention, includesantibodies that bind PCSK9 or portion thereof with a K_(D) of less thanabout 1000 nM, less than about 500 nM, less than about 300 nM, less thanabout 200 nM, less than about 100 nM, less than about 90 nM, less thanabout 80 nM, less than about 70 nM, less than about 60 nM, less thanabout 50 nM, less than about 40 nM, less than about 30 nM, less thanabout 20 nM, less than about 10 nM, less than about 5 nM, less thanabout 4 nM, less than about 3 nM, less than about 2 nM, less than about1 nM or less than about 0.5 nM, as measured in a surface plasmonresonance assay. An isolated antibody that specifically binds humanPCSK9, however, have cross-reactivity to other antigens, such as PCSK9molecules from other (non-human) species.

The anti-PCSK9 antibodies useful for the methods of the presentinvention may comprise one or more amino acid substitutions, insertionsand/or deletions in the framework and/or CDR regions of the heavy andlight chain variable domains as compared to the corresponding germlinesequences from which the antibodies were derived. Such mutations can bereadily ascertained by comparing the amino acid sequences disclosedherein to germline sequences available from, for example, publicantibody sequence databases. The present invention includes methodsinvolving the use of antibodies, and antigen-binding fragments thereof,which are derived from any of the amino acid sequences disclosed herein,wherein one or more amino acids within one or more framework and/or CDRregions are mutated to the corresponding residue(s) of the germlinesequence from which the antibody was derived, or to the correspondingresidue(s) of another human germline sequence, or to a conservativeamino acid substitution of the corresponding germline residue(s) (suchsequence changes are referred to herein collectively as “germlinemutations”). A person of ordinary skill in the art, starting with theheavy and light chain variable region sequences disclosed herein, caneasily produce numerous antibodies and antigen-binding fragments whichcomprise one or more individual germline mutations or combinationsthereof. In certain embodiments, all of the framework and/or CDRresidues within the V_(H) and/or V_(L) domains are mutated back to theresidues found in the original germline sequence from which the antibodywas derived. In other embodiments, only certain residues are mutatedback to the original germline sequence, e.g., only the mutated residuesfound within the first 8 amino acids of FR1 or within the last 8 aminoacids of FR4, or only the mutated residues found within CDR1, CDR2 orCDR3. In other embodiments, one or more of the framework and/or CDRresidue(s) are mutated to the corresponding residue(s) of a differentgermline sequence (i.e., a germline sequence that is different from thegermline sequence from which the antibody was originally derived).Furthermore, the antibodies of the present invention may contain anycombination of two or more germline mutations within the frameworkand/or CDR regions, e.g., wherein certain individual residues aremutated to the corresponding residue of a particular germline sequencewhile certain other residues that differ from the original germlinesequence are maintained or are mutated to the corresponding residue of adifferent germline sequence. Once obtained, antibodies andantigen-binding fragments that contain one or more germline mutationscan be easily tested for one or more desired property such as, improvedbinding specificity, increased binding affinity, improved or enhancedantagonistic or agonistic biological properties (as the case may be),reduced immunogenicity, etc. The use of antibodies and antigen-bindingfragments obtained in this general manner are encompassed within thepresent invention.

The present invention also includes methods involving the use ofanti-PCSK9 antibodies comprising variants of any of the HCVR, LCVR,and/or CDR amino acid sequences disclosed herein having one or moreconservative substitutions. For example, the present invention includesthe use of anti-PCSK9 antibodies having HCVR, LCVR, and/or CDR aminoacid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 orfewer, etc. conservative amino acid substitutions relative to any of theHCVR, LCVR, and/or CDR amino acid sequences disclosed herein.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-timeinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore™ system(Biacore Life Sciences division of GE Healthcare, Piscataway, N.J.).

The term “K_(D)”, as used herein, is intended to refer to theequilibrium dissociation constant of a particular antibody-antigeninteraction.

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen binding site in the variable region of anantibody molecule known as a paratope. A single antigen may have morethan one epitope. Thus, different antibodies may bind to different areason an antigen and may have different biological effects. Epitopes may beeither conformational or linear. A conformational epitope is produced byspatially juxtaposed amino acids from different segments of the linearpolypeptide chain. A linear epitope is one produced by adjacent aminoacid residues in a polypeptide chain. In certain circumstance, anepitope may include moieties of saccharides, phosphoryl groups, orsulfonyl groups on the antigen.

According to certain embodiments, the anti-PCSK9 antibody used in themethods of the present invention is an antibody with pH-dependentbinding characteristics. As used herein, the expression “pH-dependentbinding” means that the antibody or antigen-binding fragment thereofexhibits “reduced binding to PCSK9 at acidic pH as compared to neutralpH” (for purposes of the present disclosure, both expressions may beused interchangeably). For the example, antibodies “with pH-dependentbinding characteristics” includes antibodies and antigen-bindingfragments thereof that bind PCSK9 with higher affinity at neutral pHthan at acidic pH. In certain embodiments, the antibodies andantigen-binding fragments of the present invention bind PCSK9 with atleast 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, or more times higher affinity at neutral pH than atacidic pH.

According to this aspect of the invention, the anti-PCSK9 antibodieswith pH-dependent binding characteristics may possess one or more aminoacid variations relative to the parental anti-PCSK9 antibody. Forexample, an anti-PCSK9 antibody with pH-dependent bindingcharacteristics may contain one or more histidine substitutions orinsertions, e.g., in one or more CDRs of a parental anti-PCSK9 antibody.Thus, according to certain embodiments of the present invention, methodsare provided comprising administering an anti-PCSK9 antibody whichcomprises CDR amino acid sequences (e.g., heavy and light chain CDRs)which are identical to the CDR amino acid sequences of a parentalanti-PCSK9 antibody, except for the substitution of one or more aminoacids of one or more CDRs of the parental antibody with a histidineresidue. The anti-PCSK9 antibodies with pH-dependent binding maypossess, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or more histidinesubstitutions, either within a single CDR of a parental antibody ordistributed throughout multiple (e.g., 2, 3, 4, 5, or 6) CDRs of aparental anti-PCSK9 antibody. For example, the present inventionincludes the use of anti-PCSK9 antibodies with pH-dependent bindingcomprising one or more histidine substitutions in HCDR1, one or morehistidine substitutions in HCDR2, one or more histidine substitutions inHCDR3, one or more histidine substitutions in LCDR1, one or morehistidine substitutions in LCDR2, and/or one or more histidinesubstitutions in LCDR3, of a parental anti-PCSK9 antibody.

As used herein, the expression “acidic pH” means a pH of 6.0 or less(e.g., less than about 6.0, less than about 5.5, less than about 5.0,etc.). The expression “acidic pH” includes pH values of about 6.0, 5.95,5.90, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3,5.25, 5.2, 5.15, 5.1, 5.05, 5.0, or less. As used herein, the expression“neutral pH” means a pH of about 7.0 to about 7.4. The expression“neutral pH” includes pH values of about 7.0, 7.05, 7.1, 7.15, 7.2,7.25, 7.3, 7.35, and 7.4.

Preparation of Human Antibodies

Methods for generating human antibodies in transgenic mice are known inthe art. Any such known methods can be used in the context of thepresent invention to make human antibodies that specifically bind tohuman PCSK9.

Using VELOCIMMUNE™ technology (see, for example, U.S. Pat. No.6,596,541, Regeneron Pharmaceuticals) or any other known method forgenerating monoclonal antibodies, high affinity chimeric antibodies toPCSK9 are initially isolated having a human variable region and a mouseconstant region. The VELOCIMMUNE® technology involves generation of atransgenic mouse having a genome comprising human heavy and light chainvariable regions operably linked to endogenous mouse constant regionloci such that the mouse produces an antibody comprising a humanvariable region and a mouse constant region in response to antigenicstimulation. The DNA encoding the variable regions of the heavy andlight chains of the antibody are isolated and operably linked to DNAencoding the human heavy and light chain constant regions. The DNA isthen expressed in a cell capable of expressing the fully human antibody.

Generally, a VELOCIMMUNE® mouse is challenged with the antigen ofinterest, and lymphatic cells (such as B-cells) are recovered from themice that express antibodies. The lymphatic cells may be fused with amyeloma cell line to prepare immortal hybridoma cell lines, and suchhybridoma cell lines are screened and selected to identify hybridomacell lines that produce antibodies specific to the antigen of interest.DNA encoding the variable regions of the heavy chain and light chain maybe isolated and linked to desirable isotypic constant regions of theheavy chain and light chain. Such an antibody protein may be produced ina cell, such as a CHO cell. Alternatively, DNA encoding theantigen-specific chimeric antibodies or the variable domains of thelight and heavy chains may be isolated directly from antigen-specificlymphocytes.

Initially, high affinity chimeric antibodies are isolated having a humanvariable region and a mouse constant region. The antibodies arecharacterized and selected for desirable characteristics, includingaffinity, selectivity, epitope, etc, using standard procedures known tothose skilled in the art. The mouse constant regions are replaced with adesired human constant region to generate the fully human antibody ofthe invention, for example wild-type or modified IgG1 or IgG4. While theconstant region selected may vary according to specific use, highaffinity antigen-binding and target specificity characteristics residein the variable region.

In general, the antibodies that can be used in the methods of thepresent invention possess high affinities, as described above, whenmeasured by binding to antigen either immobilized on solid phase or insolution phase. The mouse constant regions are replaced with desiredhuman constant regions to generate the fully human antibodies of theinvention. While the constant region selected may vary according tospecific use, high affinity antigen-binding and target specificitycharacteristics reside in the variable region.

Specific examples of human antibodies or antigen-binding fragments ofantibodies that specifically bind PCSK9 which can be used in the contextof the methods of the present invention include any antibody orantigen-binding fragment which comprises the three heavy chain CDRs(HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region(HCVR) having an amino acid sequence selected from the group consistingof SEQ ID NOs: 2, 18, 22, 26, 42, 46, 50, 66, 70, 74, 90, 94, 98, 114,118, 122, 138, 142, 146, 162, 166, 170, 186, 190, 194, 210, 214, 218,234, 238, 242, 258, 262, 266, 282, 286, 290, 306, 310, 314, 330, 334,338, 354, 358, 362, 378, 382, 386, 402, 406, 410, 426, 430, 434, 450,454, 458, 474, 478, 482, 498, 502, 506, 522, 526, 530, 546, 550, 554,570, 574, 578, 594, 598, 602, 618, 622, 626, 642, 646, 650, 666, 670,674, 690, 694, 698, 714, 718, 722, 738 and 742, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity. The antibody or antigen-bindingfragment may comprise the three light chain CDRs (LCVR1, LCVR2, LCVR3)contained within a light chain variable region (LCVR) having an aminoacid sequence selected from the group consisting of SEQ ID NOs: 10, 20,24, 34, 44, 48, 58, 68, 72, 82, 92, 96, 106, 116, 120, 130, 140, 144,154, 164, 168, 178, 188, 192, 202, 212, 216, 226, 236, 240, 250, 260,264, 274, 284, 288, 298, 308, 312, 322, 332, 336, 346, 356, 360, 370,380, 384, 394, 404, 408, 418, 428, 432, 442, 452, 456, 466, 476, 480,490, 500, 504, 514, 524, 528, 538, 548, 552, 562, 572, 576, 586, 596,600, 610, 620, 624, 634, 644, 648, 658, 668, 672, 682, 692, 696, 706,716, 720, 730, 740 and 744, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity.

In certain embodiments of the present invention, the antibody orantigen-binding fragment thereof comprises the six CDRs (HCDR1, HCDR2,HCDR3, LCDR1, LCDR2 and LCDR3) from the heavy and light chain variableregion amino acid sequence pairs (HCVR/LCVR) selected from the groupconsisting of SEQ ID NOs: 2/10, 18/20, 22/24, 26/34, 42/44, 46/48,50/58, 66/68, 70/72, 74/82, 90/92, 94/96, 98/106, 114/116, 118/120,122/130, 138/140, 142/144, 146/154, 162/164, 166/168, 170/178, 186/188,190/192, 194/202, 210/212, 214/216, 218/226, 234/236, 238/240, 242/250,258/260, 262/264, 266/274, 282/284, 286/288, 290/298, 306/308, 310/312,314/322, 330/332, 334/336, 338/346, 354/356, 358/360, 362/370, 378/380,382/384, 386/394, 402/404, 406/408, 410/418, 426/428, 430/432, 434/442,450/452, 454/456, 458/466, 474/476, 478/480, 482/490, 498/500, 502/504,506/514, 522/524, 526/528, 530/538, 546/548, 550/552, 554/562, 570/572,574/576, 578/586, 594/596, 598/600, 602/610, 618/620, 622/624, 626/634,642/644, 646/648, 650/658, 666/668, 670/672, 674/682, 690/692, 694/696,698/706, 714/716, 718/720, 722/730, 738/740 and 742/744.

In certain embodiments of the present invention, the anti-PCSK9antibody, or antigen-binding fragment thereof, that can be used in themethods of the present invention has HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3amino acid sequences selected from SEQ ID NOs: 76/78/80/84/86/88(mAb316P) and 220/222/224/228/230/232 (mAb300N) (See US Patent App. PublNo. 2010/0166768).

In certain embodiments of the present invention, the antibody orantigen-binding fragment thereof comprises HCVR/LCVR amino acid sequencepairs selected from the group consisting of SEQ ID NOs: 2/10, 18/20,22/24, 26/34, 42/44, 46/48, 50/58, 66/68, 70/72, 74/82, 90/92, 94/96,98/106, 114/116, 118/120, 122/130, 138/140, 142/144, 146/154, 162/164,166/168, 170/178, 186/188, 190/192, 194/202, 210/212, 214/216, 218/226,234/236, 238/240, 242/250, 258/260, 262/264, 266/274, 282/284, 286/288,290/298, 306/308, 310/312, 314/322, 330/332, 334/336, 338/346, 354/356,358/360, 362/370, 378/380, 382/384, 386/394, 402/404, 406/408, 410/418,426/428, 430/432, 434/442, 450/452, 454/456, 458/466, 474/476, 478/480,482/490, 498/500, 502/504, 506/514, 522/524, 526/528, 530/538, 546/548,550/552, 554/562, 570/572, 574/576, 578/586, 594/596, 598/600, 602/610,618/620, 622/624, 626/634, 642/644, 646/648, 650/658, 666/668, 670/672,674/682, 690/692, 694/696, 698/706, 714/716, 718/720, 722/730, 738/740and 742/744.

Pharmaceutical Compositions and Methods of Administration

The present invention includes methods which comprise administering aPCSK9 inhibitor to a patient, wherein the PCSK9 inhibitor is containedwithin a pharmaceutical composition. The pharmaceutical compositions ofthe invention are formulated with suitable carriers, excipients, andother agents that provide suitable transfer, delivery, tolerance, andthe like. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. See also Powell et al.“Compendium of excipients for parenteral formulations” PDA (1998) JPharm Sci Technol 52:238-311.

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods ofadministration include, but are not limited to, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, and oral routes. The composition may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but are notlimited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen(Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis,Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark),NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (BectonDickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPENSTARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to nameonly a few. Examples of disposable pen delivery devices havingapplications in subcutaneous delivery of a pharmaceutical composition ofthe present invention include, but are not limited to the SOLOSTAR™ pen(sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (EliLilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), thePENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L. P.), andthe HUMIRA™ Pen (Abbott Labs, Abbott Park Ill.), to name only a few.

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201).In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), 1974, CRCPres., Boca Raton, Fla. In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson,1984, in Medical Applications of Controlled Release, supra, vol. 2, pp.115-138). Other controlled release systems are discussed in the reviewby Langer, 1990, Science 249:1527-1533.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by knownmethods. For example, the injectable preparations may be prepared, e.g.,by dissolving, suspending or emulsifying the antibody or its saltdescribed above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc.

Dosage

The amount of PCSK9 inhibitor (e.g., anti-PCSK9 antibody) administeredto a subject according to the methods of the present invention is,generally, a therapeutically effective amount. As used herein, thephrase “therapeutically effective amount” means a dose of PCSK9inhibitor that results in a detectable reduction (at least about 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, ormore from baseline) in one or more parameters selected from the groupconsisting of remnant cholesterol (RLP-C), VLDL-C, VLDL₁-C, VLDL₂-C,VLDL₁₊₂-C, VLDL₃-C, IDL-C, LDL₁-C, LDL₂-C, LDL₃-C, LDL₄-C, LDL₃₊₄-C.Alternatively, animal models can be used to establish whether aparticular amount of a candidate PCSK9 inhibitor is a therapeuticallyeffective amount.

In the case of an anti-PCSK9 antibody, a therapeutically effectiveamount can be from about 0.05 mg to about 600 mg, e.g., about 0.05 mg,about 0.1 mg, about 1.0 mg, about 1.5 mg, about 2.0 mg, about 10 mg,about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about70 mg, about 75 mg, about 80 mg, about 90 mg, about 100 mg, about 110mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560mg, about 570 mg, about 580 mg, about 590 mg, or about 600 mg, of theanti-PCSK9 antibody.

The amount of anti-PCSK9 antibody contained within the individual dosesmay be expressed in terms of milligrams of antibody per kilogram ofpatient body weight (i.e., mg/kg). For example, the anti-PCSK9 antibodymay be administered to a patient at a dose of about 0.0001 to about 10mg/kg of patient body weight.

Combination Therapies

The methods of the present invention, according to certain embodiments,may comprise administering a pharmaceutical composition comprising ananti-PCSK9 antibody to a patient who is on a therapeutic regimen for thetreatment of hypercholesterolemia at the time of, or just prior to,administration of the pharmaceutical composition of the invention. Forexample, a patient who has previously been diagnosed withhypercholesterolemia may have been prescribed and is taking a stabletherapeutic regimen of another drug prior to and/or concurrent withadministration of a pharmaceutical composition comprising an anti-PCSK9antibody. The prior or concurrent therapeutic regimen may comprise,e.g., (1) an agent which induces a cellular depletion of cholesterolsynthesis by inhibiting 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A(CoA) reductase, such as a statin (e.g., cerivastatin, atorvastatin,simvastatin, pitavastatin, rosuvastatin, fluvastatin, lovastatin,pravastatin, etc.); (2) an agent which inhibits cholesterol uptake andor bile acid re-absorption; (3) an agent which increase lipoproteincatabolism (such as niacin); and/or (4) activators of the LXRtranscription factor that plays a role in cholesterol elimination suchas 22-hydroxycholesterol. In certain embodiments, the patient, prior toor concurrent with administration of an anti-PCSK9 antibody is on afixed combination of therapeutic agents such as ezetimibe plussimvastatin; a statin with a bile resin (e.g., cholestyramine,colestipol, colesevelam); niacin plus a statin (e.g., niacin withlovastatin); or with other lipid lowering agents such as omega-3-fattyacid ethyl esters (for example, omacor).

Administration Regimens

According to certain embodiments of the present invention, multipledoses of a PCSK9 inhibitor (i.e., a pharmaceutical compositioncomprising a PCSK9 inhibitor) may be administered to a subject over adefined time course. The methods according to this aspect of theinvention comprise sequentially administering to a subject multipledoses of a PCSK9 inhibitor. As used herein, “sequentially administering”means that each dose of PCSK9 inhibitor is administered to the subjectat a different point in time, e.g., on different days separated by apredetermined interval (e.g., hours, days, weeks or months). The presentinvention includes methods which comprise sequentially administering tothe patient a single initial dose of a PCSK9 inhibitor, followed by oneor more secondary doses of the PCSK9 inhibitor, and optionally followedby one or more tertiary doses of the PCSK9 inhibitor.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” referto the temporal sequence of administration of the individual doses of apharmaceutical composition comprising a PCSK9 inhibitor. Thus, the“initial dose” is the dose which is administered at the beginning of thetreatment regimen (also referred to as the “baseline dose”); the“secondary doses” are the doses which are administered after the initialdose; and the “tertiary doses” are the doses which are administeredafter the secondary doses. The initial, secondary, and tertiary dosesmay all contain the same amount of the PCSK9 inhibitor, but generallymay differ from one another in terms of frequency of administration. Incertain embodiments, however, the amount of PCSK9 inhibitor contained inthe initial, secondary and/or tertiary doses varies from one another(e.g., adjusted up or down as appropriate) during the course oftreatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5)doses are administered at the beginning of the treatment regimen as“loading doses” followed by subsequent doses that are administered on aless frequent basis (e.g., “maintenance doses”).

According to exemplary embodiments of the present invention, eachsecondary and/or tertiary dose is administered 1 to 26 (e.g., 1, 1½, 2,2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½,12, 12½, 13, 13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½,20, 20½, 21, 21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½, or more)weeks after the immediately preceding dose. The phrase “the immediatelypreceding dose,” as used herein, means, in a sequence of multipleadministrations, the dose of antigen-binding molecule which isadministered to a patient prior to the administration of the very nextdose in the sequence with no intervening doses.

The methods according to this aspect of the invention may compriseadministering to a patient any number of secondary and/or tertiary dosesof a PCSK9 inhibitor. For example, in certain embodiments, only a singlesecondary dose is administered to the patient. In other embodiments, twoor more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses areadministered to the patient. Likewise, in certain embodiments, only asingle tertiary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiarydoses are administered to the patient.

In embodiments involving multiple secondary doses, each secondary dosemay be administered at the same frequency as the other secondary doses.For example, each secondary dose may be administered to the patient 1 to2, 4, 6, 8 or more weeks after the immediately preceding dose.Similarly, in embodiments involving multiple tertiary doses, eachtertiary dose may be administered at the same frequency as the othertertiary doses. For example, each tertiary dose may be administered tothe patient 1 to 2, 4, 6, 8 or more weeks after the immediatelypreceding dose. Alternatively, the frequency at which the secondaryand/or tertiary doses are administered to a patient can vary over thecourse of the treatment regimen. The frequency of administration mayalso be adjusted during the course of treatment by a physician dependingon the needs of the individual patient following clinical examination.

The present invention includes administration regimens comprising anup-titration option (also referred to herein as “dose modification”). Asused herein, an “up-titration option” means that, after receiving aparticular number of doses of a PCSK9 inhibitor, if a patient has notachieved a specified reduction in one or more defined therapeuticparameters, the dose of the PCSK9 inhibitor is thereafter increased. Forexample, in the case of a therapeutic regimen comprising administrationof 75 mg doses of an anti-PCSK9 antibody to a patient at a frequency ofonce every two weeks, if after 8 weeks (i.e., 5 doses administered atWeek 0, Week 2 and Week 4, Week 6 and Week 8), the patient has notachieved a serum LDL-C concentration of less than 70 mg/dL, then thedose of anti-PCSK9 antibody is increased to e.g., 150 mg administeredonce every two weeks thereafter (e.g., starting at Week 10 or Week 12,or later).

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1. Generation of Human Antibodies to Human PCSK9

Human anti-PCSK9 antibodies were generated as described in U.S. Pat. No.8,062,640. The exemplary PCSK9 inhibitor used in the following Exampleis the human anti-PCSK9 antibody designated “mAb316P” (also referred toin the scientific literature as “alirocumab”). mAb316P has the followingamino acid sequence characteristics: heavy chain variable region (HCVR)comprising SEQ ID NO:90; light chain variable domain (LCVR) comprisingSEQ ID NO:92; heavy chain complementarity determining region 1 (HCDR1)comprising SEQ ID NO:76; HCDR2 comprising SEQ ID NO:78; HCDR3 comprisingSEQ ID NO:80; light chain complementarity determining region 1 (LCDR1)comprising SEQ ID NO:84; LCDR2 comprising SEQ ID NO:86; and LCDR3comprising SEQ ID NO:88.

Example 2: Dynamics Between Anti-PCSK9 Antibody Levels and Low-DensityLipoprotein Cholesterol (LDL-C) Levels

Introduction

Descriptive analyses of pharmacokinetic and pharmacodynamic data fromPhase I and II studies of mAb316P were conducted to characterizerelationships between mAb316P, PCSK9 and LDL-C levels. Three differentclinical trials were included in this analysis:

(1) Single-Dose Study:

Phase I, single-dose study in healthy subjects not receiving backgroundstatin therapy in which subjects received single subcutaneous (SC) dosesof mAb316P 50, 100, 150, and 250 mg, or placebo.

(2) Multiple-Dose Study:

A Phase I study in cohorts of heterozygous familial hypercholesterolemia(HeFH) and non-familial hypercholesterolemia (FH) patients receivingatorvastatin therapy and non-FH patients receiving diet alone. Patientsreceiving atorvastatin were randomized to mAb316P (50, 100, or 150 mg)or placebo administered SC on Days 1, 29, and 43. Non-FH patientsreceiving diet alone were randomized to mAb316P 150 mg or placeboadministered SC on Days 1 and 29.

(3) Phase II Study:

A 12-week study in hypercholesterolemic patients receiving atorvastatintherapy: patients randomized to mAb316P 50, 100, or 150 mg SC every 2weeks (Q2W), 200 or 300 mg SC every 4 weeks (Q4W), or placebo.

Results

Single administration of mAb316P 150 mg SC rapidly bound to and rapidlyreduced circulating free PCSK9 levels; this was followed by a drop inLDL-C. The return of free PCSK9 towards starting levels was noted toprecede the return of LDL-C levels (FIG. 1). Free PCSK9 levels reachednadir by Day 3 following administration of mAb316P 50, 100, or 150 mg SCin HeFH or non-FH patients (FIG. 2). The return of free PCSK9 tostarting levels was slower in the patients who received mAb316P plusdiet alone versus patients who received mAb316P plus atorvastatin (FIG.2). The greatest mean LDL-C reductions were recorded on Day 8 for the 50mg and 100 mg doses and Day 15 for the 150 mg dose; this delay relativeto free PCSK9 concentrations may possibly be due to the time requiredfor the production of new LDL-C receptors. A representative hysteresiscurve showing the relationship between total mAb316P and LDL-C followingadministration of the 150 mg dose is shown in FIG. 3.

Higher baseline free PCSK9 levels were observed in patients takingstatins as compared with diet alone (FIG. 4). There was increasedtarget-mediated clearance of mAb316P with concurrent statin therapy (dueto higher free PCSK9 levels, likely reflective of an increase inproduction rate) compared with mAb316P and diet therapy alone. Thisappeared to affect the duration of LDL-C lowering over a 4-week dosinginterval (Days 1 to 29), which was reduced with concurrent statintherapy versus diet only; however, this effect was not seen over the2-week dosing interval (Days 29 to 43) (FIG. 5).

In hypercholesterolemic (non-FH) patients receiving concomitant statintherapy, mAb316P Q2W resulted in uniformly strong LDL-C lowering at 12weeks. Patients on Q2W dosing regimens exhibited lower variability inthe LDL-C response at Week 12 versus Q4W dosing (FIG. 6); coefficient ofvariation 16% versus 49% for 150 mg Q2W and 300 mg Q4W, respectively.

Conclusions

Dynamics were apparent between mAb316P, free PCSK9 and LDL-C levels.mAb316P treatment resulted in reductions in free PCSK9 levels within 3days of dosing and peak reductions in LDL-C₈₋₁₅ days after dosing.

The clearance of mAb316P was accelerated by its binding to free PCSK9(target-mediated clearance). Lower levels of free PCSK9 resulted in alonger duration of efficacy through lower target-mediated clearance.Previous studies have shown that administration of statins increases theproduction of free PCSK9. In the studies analyzed herein, patients whoreceived mAb316P plus diet alone had lower baseline free PCSK9 levelsand higher mAb316P blood concentrations compared with patients whoreceived mAb316P plus atorvastatin therapy.

In patients receiving concomitant statin therapy, mAb316P administeredQ2W achieved lower variation in LDL-C lowering at the end of the dosinginterval as compared with Q4W dosing regimens. In Q4W dosing,patient-to-patient differences in target-mediated clearance (among otherfactors) appeared to contribute to differences in the duration ofmaximum LDL-C between Weeks 2 and 4 post dose and therefore widerdifferences in achieved efficacy 4 weeks after dosing. These differenceswere not observed when doses were administered Q2W.

Example 3A: An Anti-PCSK9 Antibody Reduces Cholesterol Concentrations ofSerum Remnant Lipoprotein Fractions, Very Low Density Lipoproteins,Triglycerides, and Lipoprotein(a) [Lp(a)]

Introduction

Increased very low-density lipoproteins (VLDL) levels form part of apattern of atherogenic dyslipidemia, which predisposes to prematureatherosclerosis. Remnant lipoproteins are products of VLDL lipolysis,and include VLDL₃ and intermediate-density lipoproteins (IDL, the directprecursor to LDL formation).

Lipoprotein(a) [Lp(a)] consists of apolipoprotein(a) [apo(a)] covalentlybound to the apo B component of a cholesterol-rich lipoprotein which isapproximately the same size as a low-density lipoprotein (LDL) particle.Lp(a) metabolism is under mostly genetic regulation, in that more than90% of its plasma concentration is influenced by quantitative andqualitative polymorphisms at the apo(a) gene (LPA). Elevated Lp(a)levels are considered to be an independent risk factor forcardiovascular disease, with risk continuing to increase as levels rise.Although elevated Lp(a) may promote atherosclerosis through intimaldeposition of cholesterol, it may also promote thrombosis due to a highhomology with plasminogen, although it lacks protease activity. Inpatients undergoing coronary angiography, Lp(a) concentrations>30 mg/dLhave been shown to be associated with an increased risk of angiographicstenosis and major coronary events. Furthermore, Lp(a) levels andgenotype have been shown to be associated with aortic-valvecalcification, suggesting it may play a role in causation.

The objective of this Example was to test the hypothesis that mAb316P, afully human monoclonal antibody to proprotein convertasesubtilisin/kexin type 9 (PCSK9), reduces serum levels of VLDL andlipoprotein remnants, contributing to its ability to reduce serum LDL-Cand non-HDL-C, and to confirm the Lp(a) lowering activity of mAb316P inpatients.

Three multicenter, double-blind, parallel-group, placebo-controlledtrials were conducted in patients with primary hypercholesterolemia(Study A, n=183; Study B, n=92) or heterozygous familialhypercholesterolemia (Study C, n=77). A summary of the designs anddosing for the three trials is shown in Table 1.

TABLE 1 Summary of Designs and Dosing for mAb316P Clinical Trials StudyA B C Duration 12 weeks 8 weeks 12 weeks Patients hypercholesterolemiahypercholesterolemia heterozygous familial (n = 183) (n = 92)hypercholesterolemeia (n = 77) mAb316P Doses 200-300 mg Q4W 150 mg Q2W +ATV 10 150, 200, 300 mg Q4W  50-100 mg Q2W to 80 mg 150 mg Q2W    150 mgQ2W 150 mg Q2W + ATV 10 (n = 16) (n = 31) to 80 mg (total n = 61)Placebo n = 31 Placebo + ATV 10 n = 15 to 80 mg (n = 31) Pooled AnalysisPopulation mAb316P 150 mg Q2W (n = 108) Placebo (n = 77)

In Study B, all patients received atorvastatin (ATV) 10 mg prior torandomization, which was uptitrated to ATV 80 mg at the start ofrandomized treatment in the placebo and one mAb316P arm.

Patients on background atorvastatin or statins+/−ezetimibe receivedmAb316P 50-300 mg administered subcutaneously (SC) either every 2 or 4weeks (Q2W, Q4W), depending on the study. The mAb316P 150 mg Q2W dosewas common to all three trials.

In post hoc analyses, lipoproteins were subfractionated by vertical autoprofile (VAP) testing. The VAP method is a single, directultracentrifugation test that separates the lipoprotein fractionsaccording to their densities in a vertical rotor, which allows highresolution of each lipoprotein class and subclass. The bottom of thetube is punctured and the cholesterol in each layer is measured with aspectrophotometer after the addition of an enzymatic cholesterolreagent. Percent changes in VLDL-C, VLDL₁₊₂-C (a measure of cholesterolin “large, buoyant” VLDL particles), triglycerides (TG), VLDL₃-C, IDL-Cand total remnant lipoprotein cholesterol levels (RLP-C; VLDL₃-C+IDL-C)in patients treated with mAb316P 150 mg Q2W vs. placebo were analyzed atweek 12 (Study A), week 8 (Study B) and week 6 (Study C) using ANCOVA.

In addition, baseline and on-treatment Lp(a) levels were assessed inpatients from the three different Phase 2 studies receiving mAb316P 150mg Q2W or placebo. In all Phase 2 studies Lp(a) levels were measured atthe same laboratory and using the same method. Data on Lp(a) levels atbaseline and end of treatment (Week 8/12 on-treatment value or the lastavailable on-treatment value carried forward) from the modifiedintention-to-treat populations of the three studies were pooled, andpercentage changes from baseline for mAb316P 150 mg Q2W and placebo werecompared using analysis of covariance with treatment group and study asfixed effects and baseline Lp(a) as covariate. P-values associated withthese exploratory analyses are provided for descriptive purposes onlyand were not adjusted for multiplicity. The relationship between thepercentage changes from baseline in Lp(a) and LDL-C was assessed usinglinear regression, and the Spearman's correlation coefficient wascalculated.

Results/Conclusions

In the 3 studies, reductions in TG, VLDL-C, and in the cholesterolcontent of remnant lipoprotein were observed with mAb316P vs. placebo.Results are summarized in Table 2 and FIGS. 7-9.

TABLE 2 Baseline (mg/dL) Endpoint (mg/dL) Percent Change p-value (s)VLDL 23.24 to 26.31 16.62 to 18.12 −22.33 to −27.95 0.0023 to <0.0001VLDL₁₊₂-C  9.79 to 10.59 6.59 to 7.31 −21.87 to −31.45 0.0178 to <0.0001VLDL₃ 13.55 to 15.62 10.03 to 10.88  −21.9 to −26.66 0.0011 to <0.0001TG 135.72 to 157.19  99.9 to 124.44 −13.07 to −21.19 0.6945 to 0.0003  IDL 15.38 to 22.06 6.79 to 8.62 −50.28 to −55.75 <0.0001 RLPC 29.34 to37.69 16.86 to 19.5  −37.05 to −44.04 <0.0001

The entire spectrum of atherogenic lipoproteins (LDL-C, IDL-C, VLDL-Cand RLP subfractions) was reduced by mAb316P, against a background oflipid lowering therapy (statins±ezetimibe), in heFH/non-FH patients.This Example therefore illustrates that mAb316P significantly reducedserum TG and VLDL-C, in addition to significant reductions incholesterol concentrations of remnant lipoprotein fractions separable byVAP, including VLDL₃-C and IDL-C.

Baseline and on-treatment Lp(a) data were available for 102 of the 108patients who received mAb316P 150 mg Q2W and 74 of the 77 patients whoreceived placebo in the Pooled Analysis Population. Baseline values areshown in Table 3.

TABLE 3 Baseline Lp(a) Levels in Patients Included in the PooledAnalysis Placebo mAb316P 150 mg Q2W (n = 74) (n = 102) Lp(a) values inmg/dL Overall Population, median 19.0 (6.0-77.0) 29.5 (8.0-70.0) (IQR)Range 1.5-299.0 1.5-181.0 Number of Patients Subdivided by BaselineLp(a) ≤50 mg/dL, n (%) 49 (66.2) 68 (66.7) >50 mg/dL, n (%) 25 (33.8) 36(35.3) *Patients from the mITT population with Lp(a) data available atbaseline and end of treatment (Week 8/12 on-treatment value or the lastavailable on-treatment value carried forward).

As shown in Table 3, 36 (35%) patients treated with mAb316P and 25 (33%)patients treated with placebo had baseline Lp(a) >50 mg/dL, consideredas the high-risk cut-point by the EAS guidelines.

Absolute and percentage median reductions from baseline in Lp(a) aresummarized in Table 4.

TABLE 4 Lp(a) Change From Baseline Patients Subdivided by Baseline Lp(a)All Patients ≤50 mg/dL >50 mg/dL pooled pooled pooled pooled pooledpooled placebo mAb316P placebo mAb316P placebo mAb316P treated treatedtreated treated treated treated (n = 74) (n = 102) (n = 49) (n = 68) (n= 25) (n = 34) Median Lp(a) Change From Baseline, mg/dL (IQR; LOCF) −0.5−9.0 0.0 −3.5 −5.0 −26.5 (−5.0-2.0) (−19.0-−2.0)* (−3.0-1.5)(−11.5-−1.5)* (−11.0-6.0) (−39.0-−16.0)* Median Lp(a) Percent ChangeFrom Baseline, (IQR; LOCF) −0.3% −30.3% 0.0% −36.1% −4.4% −27.0%(−16.7-11.5) (−50.0-−19.4)* (−16.7-16.7) (−51.1-−17.4)* (−9.4-7.1)(−32.6-−19.6)* *P < 0.0001 vs placebo.

As shown in Table 4, the median percentage reduction from baseline inLp(a) was −30.3% with mAb316P 150 mg Q2W versus −0.3% with placebo(P<0.0001). The absolute median reductions in Lp(a) from baseline weresubstantially greater in patients with the higher baseline Lp(a). Insummary, an analysis of data pooled from three Phase 2 trials conductedwith mAb316P 150 mg Q2W demonstrated significant reductions in Lp(a)versus placebo, including in patients with baseline Lp(a)>50 mg/dL. Inthose patients considered at higher cardiovascular disease risk due toelevated Lp(a), the percentage reductions in Lp(a) appeared to be ofsimilar magnitude resulting in greater absolute reductions in Lp(a).

Example 3B: An Anti-PCSK9 Antibody Reduces Apolipoprotein CII and CIIILevels in Serum

Introduction

Apoprotein (apo) CIII inhibits lipoprotein lipase (LPL)-mediatedcatabolism of VLDL triglycerides. Apo CII appears to have a more complexrelationship with VLDL and LPL activity that may depend on baselinetriglyceride levels. Apo CII is, in general, an important activator ofLPL

LPL hydrolyzes the triglyceride mass of VLDL and its remnants. In thisExample, the ability of an anti-PCSK9 antibody, mAb316P, to reduceVLDL-C and remnants by impacting serum levels of apoproteins CII andCIII was investigated.

Three multicenter, double-blind, parallel-group, placebo-controlledtrials were conducted in patients with primary (non-familial)hypercholesterolemia (non-FH) (Study A, n=183; Study B, n=92) orheterozygous familial hypercholesterolemia (heFH) (Study C, n=77).Patients (who were receiving atorvastatin or statins+/−ezetimibe) weretreated with mAb316P 50-150 mg every 2 wks (Q2W) or 150-300 mg every 4wks (Q4W) depending on the study. The mAb316P 150 mg Q2W dose was commonto all three trials. (See Example 3A, Table 1).

In post hoc analyses, apo CII and CIII concentrations were measured viaimmunoassay. (using reagent kits from Randox Laboratories Limited, UK[apo CII, Cat. No. LP3866; apo CIII, Cat. No. LP3865] and an ArchitectCi8200 analyzer [Abbott Laboratories, IL]). The immunoassay methods werebased on the reaction of a sample containing human apo CII (or apo CIII)and specific antiserum to apo CII (or CIII) to form an insoluble complexwhose concentration can be measured turbidimetrically at 340 nm. Bothassays were validated for analytical performance.

Percent change from baseline in apo CII and apo CIII in the placebogroup was compared with patients treated with mAb316P, analyzed at week12 (Study A), week 8 (Study B) and week 6 (Study C) using analysis ofcovariance. Study C had a Week 12 endpoint; however, 6-week data wereused due to reduced number of patients with available stored samples at12 weeks (n=17) compared with 6 weeks (n=75).

Results

Percent change in apo CII and CIII from baseline by treatment group formAb316P 150 mg Q2W in Studies A, B and C are shown in Table 5. Valuesshown are mean (SD), except for apo CIII, where values are median (IQR).Data are from Week 12 (study A), Week 8 (study B) and Week 6 (study C).FIG. 13 shows the dose response changes in apo CII (panel A) and CIII(panel B) for study A.

TABLE 5 P-value P-value Apo CII versus Apo CIII versus % change placebo% change placebo Study A Placebo 12.0 3.76 (33.0) (−5.5 to 27.6) mAb316P−21.4 <0.00001 −24.7 <0.00001 150 mg (26.5) (−28.4 to −2.9) Q2W Study BPlacebo −10.6 −7.0 (30.2) (29.8 to 7.3) mAb316P −10.0 0.98 −4.9 0.4426150 mg Q2W + (22.7) (−24.4 to 1.0) atorvastatin 10 mg mAb316P −29.30.0067 −22.9 0.0165 150 mg Q2W + (19.29) (−28 to −14.0) atorvastatin 80mg Study C Placebo 1.6 −3.3 (15.5) (−15.3 to 5.6) mAb316P −9.4 0.0794−20.2 0.019 150 mg (28.1) (−29.0 to 2.5) Q2W

mAb316P reduced apo CII and CIII at all doses, but a clear dose responsewas observed (Table 5, FIG. 13). In all three studies, mAb316P reducedapo CII in patients from baseline (Table 5). The mean reductions frombaseline at Week 12 (study A), Week 8 (study B) and Week 6 (study C)were 21.4% (P<0.00001), 29.3% (P=0.0067) and 9.4% (P=0.08), respectively(P-values compared with placebo). Apo CIII was reduced by 24.7%(P<0.00001), 22.9% (P=0.017) and 20.2% (P=0.019), for studies A, B andC, respectively (P-values compared with placebo).

Conclusions

Therapy with anti-PCSK9 antibody mAb316P was associated with significantreductions in serum apo CII and CIII. There was a nearly 1:1 reductionin both apo CII and CIII in patients with non-FH. In patients with heFH,apo CIII decreased approximately two-fold more than apo CII. Thereductions in apo CII and CIII may be a manifestation of eitherincreased clearance or reduced production/secretion of VLDL particles,known carriers of these apoproteins.

Example 4: An Anti-PCSK9 Antibody Reduces Cholesterol Concentrations ofall Serum Low-Density Lipoprotein Fractions

Introduction

Low-density lipoprotein cholesterol (LDL-C) is composed of a continuumof LDL particles of different densities and states of lipidation.Analysis of effects of lipid-modifying therapies on LDL particlecomposition may aid understanding of treatment mode-of-action. Thepurpose of this Example was to test the hypothesis that mAb316P, a fullyhuman monoclonal antibody to proprotein convertase subtilisin/kexin 9(PCSK9), reduces LDL-C by decreasing multiple LDL fractions.

Three multicenter, double-blind, parallel-group, placebo-controlledtrials were conducted in patients with primary hypercholesterolemia(Study A, n=183; Study B, n=92) or heterozygous familialhypercholesterolemia (Study C, n=77). Patients (who were receivingatorvastatin or statins+/−ezetimibe) were treated with mAb316P 50-150 mgevery 2 wks (Q2W) or 150-300 mg every 4 wks (Q4W) depending on thestudy. The mAb316P 150 mg Q2W dose was common to all three trials. (SeeExample 3A, Table 1).

In post hoc analyses, lipoproteins were subfractionated by vertical autoprofile (VAP) testing. Percent change in LDL₁-C (“large buoyant” LDL),LDL₂-C, LDL₃-C, and LDL₄-C (“small, dense” LDL) levels in patientsreceiving mAb316P 150 mg Q2W vs. placebo from baseline to week 12 forStudy A, week 8 for Study B, and week 6 for Study C were assessed usingANCOVA. The LDL₃-C and LDL₄-C fractions were analyzed individually andin a pooled analysis. This was done because LDL₄-C typically exists atsubstantially lower concentrations than LDL³⁻⁴-C. There was much moresubstantial variation in percent reductions in LDL₄-C. The sum ofLDL₃₊₄-C represents a sum of the two smallest and densest LDL-Cfractions.

Results/Conclusions

Significant reductions from baseline in cholesterol content of all LDLsubfractions separable by VAP were observed in patients receivingmAb316P 150 mg Q2W in the 3 studies, vs. placebo. Results are summarizedin Table 6 and FIGS. 10-12.

TABLE 6 Baseline (mg/dL) Endpoint (mg/dL) Percent Change p-value LDL₁16.18 to 25.92 3.56 to 6.81 −68.87 to −77.84 <0.0001 LDL₂ 20.29 to 31.463.04 to 7.84 −77.85 to −84.17 <0.0001 LDL₃₄ 46.33 to 49.16  14.9 to22.29 −48.77 to −68.49 <0.0001

This Example therefore illustrates that therapy with a monoclonalantibody directed against PCSK9 (mAb316P) significantly reducedcholesterol concentrations in LDL fractions separable by VAP. Thepreviously demonstrated LDL-C reductions with mAb316P were confirmedacross the spectrum of LDL-C sub-fractions.

Example 5: Effects of an Anti-PCSK9 Antibody on Lipoprotein ParticleConcentrations

Background

The cholesterol content of low-density lipoprotein particles (LDL-P) andhigh-density lipoprotein particles (HDL-P) can vary greatly amongindividuals. For example, one individual may have larger,cholesterol-rich LDL-P, and another will have smaller, cholesterol-poorLDL-P. Therefore measurements of serum levels of low-density lipoproteincholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C)often do not correlate with circulating LDL-P and HDL-P number.

Epidemiological studies have demonstrated that atheroscleroticcardiovascular disease risk tracks with both LDL-C and LDL-P. However,in patient populations where these measures are discordant (e.g.,patients with diabetes or metabolic syndrome), risk has been shown totrack more consistently with LDL-P than LDL-C. Consequently, multipleexpert panels and consensus statements have advocated the adjunctive useof LDL-P as a target of therapy in the management of high-risk patients.

Nuclear magnetic resonance (NMR) technology can accurately measure thenumber of circulating lipoprotein particles to identify patients withdiscordantly high LDL-P levels that require intensification of therapy.

The anti-PCSK9 antibody mAb316P, has been shown to reduce LDL-C by up to72.4% in patients on stable doses of atorvastatin over 12 weeks withsimilar rates of adverse events between placebo and mAb316P-treatmentgroups. mAb316P reduced apolipoprotein B by up to 56% proportionallywith the changes in LDL-C. The effect of mAb316P on lipoprotein particlenumbers in hypercholesterolemic patients has heretofore not beenreported.

The present Example sets forth an evaluation of the effects of mAb316Pon the concentration and size of lipoprotein particles.

Methods

A sub-study of a randomized double-blind Phase II trial was carried outwith respect to patients with LDL-C ≥100 mg/dL who received placebo(n=31) or mAb316P 150 mg administered subcutaneously (SC) every 2 weeks(Q2W) (n=28) on top of stable daily atorvastatin (10, 20 or 40 mgdaily). Lipid and lipoprotein tests were performed using samplescollected after a 12-hour overnight fast. Lipoprotein particle profileswere measured by NMR spectroscopy using the LipoProfile-3 algorithm atLipoScience, Inc. (Raleigh, N.C.). LDL-P and HDL-P subclasses werequantified from the amplitudes of their spectroscopically distinct lipidmethyl group NMR signals, and weighted-average LDL and HDL sizes werederived from the sum of the diameter of each subclass multiplied by itsrelative mass percentage based on the amplitude of its methyl NMRsignal.

Diameter range estimates for the subclasses were as follows:

VLDL-P: small VLDL-P=29 to 42 nm; medium VLDL-P=42 to 60 nm; largeVLDL-P>60 nm;

LDL-P: small LDL-P=18 to 20.5 nm; large LDL-P=20.5 to 23 nm;intermediate-density lipoprotein particles (IDL-P)=23 to 29 nm;

HDL-P: small HDL-P=7.3 to 8.2 nm; medium HDL-P=8.2 to 9.4 nm; and largeHDL-P=9.4 to 14 nm.

Total VLDL-P, LDL-P, and HDL-P equal the sum of the particle numberconcentrations of the VLDL, LDL, and HDL subclasses, respectively.Endpoints for the analysis were percentage change in the concentrationof LDL-P, VLDL-P and HDL-P from baseline to Week 12.

Any variables that were not normally distributed were transformed forall statistical tests, but the untransformed variables are presented inthe tables herein. Mean (standard deviation [SD]) are reported forcontinuous normally distributed variables, while median (interquartilerange [IQR]) are reported for any non-normally distributed variables.Count and percentage are reported for categorical variables. Todetermine if the treatment group was significantly different thanplacebo, Fisher's exact tests were performed and the associated P-valuesare reported. To determine if each of the treatment groups weresignificantly different than placebo, t-tests were performed and theassociated P-values are reported. To determine if there was asignificant difference in percent change between baseline and Week 12for the placebo compared to the treatment groups analysis of covarianceswere performed with the baseline value as a covariate.

Results

Table 7 shows lipoprotein particle concentrations before and aftermAb316P 150 mg SC Q2W compared to placebo.

TABLE 7 Lipoprotein particle concentrations (nmol/L) for placebo (n =31) and mAb316P 150 mg Q2W (n = 28). Mean (sd) or median (Q1:Q3)[proportion of total] Baseline Week 12 % change PLACEBO LDL Total LDL1422.5 (321.3) 1383.8 (327.9)  −1.0% Intermediate 110 (51:166.5) 57(24.5:144.5) −15.0% density lipoprotein Large LDL 546.6 (205.3) 431.8(217.4) −21.8% Small LDL 755.3 (304.9) 847.6 (375.1)   17.8% VLDL +Total VLDL + 61.9 (47.8:95.6) 83.9 (45:102.2)   33.4% chylomicronchylomicron Large VLDL + 3.5 (2.1:8.5) 4.3 (1.8:9.4)   14.3% chylomicronMedium VLDL 19.3 (13.1:33.9) 33.1 (13.1:51.9)   24.0% Small VLDL 35.3(28.2:47.8) 37.3 (23.4:56.3)   21.4% HDL Total HDL 32.9 (6.4) 33.2 (7.4)   1.4% Large HDL 3.8 (2.0) 3.9 (2.2)   6.99% Medium HDL 9.2 (5.9:14.4)8 (5.4:10.2) −13.9% Small HDL 18.8 (5.3) 21.3 (5.8)   18.4% mAb316P 150mg Q2W LDL Total LDL 1320 (304.0) 475.4 (167.3)†  −63.3%† Intermediate84.5 (33:115) 37 (12:66)* −52.8% density lipoprotein Large LDL 532.2(212.9) 152.4 (107.6)† −71.3% Small LDL 666.5 (333.8) 279.9 (191.0)†−54.0% VLDL + Total VLDL + 71.5 (36.9:94.1) 42.0 (30.5:54.1)†  −36.4%†chylomicron chylomicron Large VLDL + 3.6 (1.8:8.5) 3.1 (1.7:6.9)   5.37%chylomicron Medium VLDL 18.3 (10.7:50) 14.4 (8.2:26.9)† −38.92%  SmallVLDL 35.7 (25:49.5) 21.4 (19.9:26.7)* −33.4% HDL Total HDL 32.6 (6.3)36.1 (6.5)*  11.2%* Large HDL 4.8 (3.1) 6.1 (3.5)*   44.6% Medium HDL7.8 (5.6:10.7) 9.8 (6.6:11.3) 17.65% Small HDL 19.4 (4.1) 20.0 (5.7)*   2.8% *P < 0.05; †P < 0.001 vs. placebo

mAb316P reduced mean LDL-P by 63% vs 1% for placebo (P<0.0001) andmedian VLDL-P by 36% vs an increase of 33% for placebo (P<0.0001). HDL-Plevels increased 11% for mAb316P vs 1% for placebo (P<0.05). Changes inall particle subclasses were directionally similar.

Conclusion

mAb316P significantly reduced LDL-P and other lipoprotein particles in amanner similar to its previously reported effect on LDL-C andapolipoprotein B.

Example 6: Effects of an Anti-PCSK9 Antibody on Lipoprotein Subfractionsas Determined by Ion Mobility

Background

mAb316P (also known as Alirocumab) has demonstrated significantreductions in low-density lipoprotein cholesterol (LDL-C) in Phase 2clinical trials. LDL-C tracks closely with cardiovascular disease (CVD)risk and subfractions of LDL, defined by differences in particle sizeand density, have also been associated with varying degrees of CVD risk.Analysis of lipoprotein subfractions may provide further insight intoCVD risk evaluation and the effects of lipid-lowering treatments seen inindividual patients.

Methods

A Phase 2 randomized, double-blind, clinical trial was conducted inpatients with hypercholesterolemia and LDL-C levels≥100 mg/dL, in which31 patients received placebo and 27 patients received alirocumab 150 mgevery 2 weeks (Q2W) via a 1-mL subcutaneous auto-injection in additionto stable atorvastatin (10-40 mg daily). In the present sub-study,lipoprotein subfractions were determined at baseline and at Week 12using ion mobility.

Results

Changes in lipoprotein subfractions and lipid parameters from baselineto Week 12 are set forth in Tables 8A (placebo-treated) and 8B(mAb316P-treated).

TABLE 8A Placebo Percent Baseline Week 12 Change Lipoproteinsubfractions VLDL + IDL + 1648.44 (453.5) 1728.88 (434.58) 8.25 TotalLDL VLDL (large + 134.97 (109.01 to 152.85) 140.48 (106.18 to 193.49)6.64 intermediate + small) IDL (IDL 1 + 2) 314.28 (282.74 to 407.95)303.35 (277.99 to 376.17) −3.3 Total LDL 1172.99 (362.85) 1233.76(331.13) 8.8 Large LDL 448.23 (173.52) 419.36 (160.98) −0.26 (LDL 1 +2a) Medium LDL 233.2 (105.34) 228.92 (88.12) 5.07 (LDL 2b) Small LDL195.17 (85.53) 225.98 (105.1) 24.39 (LDL 3a) Very Small LDL 296.4(91.75) 359.49 (136.92) 24.61 (LDL 4a + 4b + 4c + 3b) HDL (HDL 2b;23532.48 (5392.57) 24400.97 (5798.21) 0.06 HDL3 + 2a) Lipid parametersLDL-C, mg/dL 130.2 (27.3) 120.5 (27.0) −5.1 HDL-C, mg/dL 49.0 (10.3)48.9 (13.2) −1.0 TG, mg/dL 124.0 (92.0 to 187.5) 127.0 (98.0 to 197.0)9.7 Apo-B, mg/dL 108.3 (19.3) 109.2 (27.0) 2.2 Apo-A1, g/L 1.4 (1.3 to1.6) 1.4 (1.3 to 1.7) 0.0 Lp(a), g/L 0.2 (0.1 to 0.9) 0.2 (0.07 to 0.85)0.0

TABLE 8B mAb316P 150 mg Q2W Percent Change Baseline Week 12 (p-value)Lipoprotein subfractions VLDL + IDL + Total LDL 1542.82 (435.5) 767.78(212.39) −48.68 (<0.0001) VLDL (large + 138.17 (96.41 to 167.8) 77.2(51.81 to 95.8) −51.37 intermediate + small) (<0.0001) IDL (IDL 1 + 2)309.64 (268.32 to 363.95) 155.19 (134.05 to 194.5) −52.29 (<0.0001)Total LDL 1078.28 (347.61) 529.35 (171) −49.84 (<0.0001) Large LDL (LDL1 + 2a) 421.08 (130.03) 152.66 (53.4) −62.66 (<0.0001) Medium LDL (LDL2b) 200.86 (91.4) 78.67 (40.76) −58.69 (<0.0001) Small LDL (LDL 3a)172.02 (104.89) 79.24 (38.36) −48.56 (<0.0001) Very Small LDL (LDL284.31 (101.87) 218.77 (55.66) −17.91 4a + 4b + 4c + 3b) (<0.0001) HDL(HDL 2b; 23480.25 (5281.63) 21888.87 (4515.15) −0.05 HDL3 + 2a) (0.0339)Lipid parameters LDL-C, mg/dL 123.9 (26.7) 34.2 (15.6) −72.4 (<0.0001)*HDL-C, mg/dL 53.3 (16.1) 55.1 (14.8) 5.5 (0.570)‡ TG, mg/dL 140.5 (92.5to 177.5) 99.0 (79.0 to 139.0) −18.9 (0.0006)‡ Apo-B, mg/dL 101.6 (26.6)44.1 (14.1) −56.1 (<0.0001)‡ Apo-A1, g/L 1.5 (1.3 to 1.7) 1.6 (1.4 to1.7) 1.4 (0.1524)‡ Lp(a), g/L 0.3 (0.1 to 0.6) 0.1 (0.05 to 0.41) −28.6(<0.0001)‡ *Statistically significant p-value according to thehierarchical procedure. ‡P-values are not adjusted for multiplicity andare for descriptive purposes only.

Tables 8A and 8B illustrate changes in lipoprotein subfractions andlipid parameters in placebo and mAb316P 150 mg Q2W groups, respectively,from baseline to Week 12. Compared with placebo, mAb316P reduced verylow-density lipoprotein (VLDL)+intermediate-density lipoprotein(IDL)+total LDL by 48.7% (p<0.0001), VLDL by 51.4% (p<0.0001), IDL by52.3% (p<0.0001) and total LDL by 49.8% (p<0.0001); changes in othersubfractions were directionally similar. No significant change was notedfor HDL. There was a highly significant difference in percent changereduction in very small LDL versus the other LDL fractions (p<0.0001).

Conclusions

As determined by ion mobility, mAb316P significantly reduced LDLsubfractions when added to stable atorvastatin therapy, similar to itspreviously reported effect on LDL-C.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

What is claimed is:
 1. A method for reducing in a patient the serumconcentration of IDL-C; the method comprising selecting a patient withelevated serum IDL-C, and administering to the patient a pharmaceuticalcomposition comprising a PCSK9 inhibitor; wherein the patient isotherwise healthy except for exhibiting elevated serum IDL-C; andwherein the PCSK9 inhibitor is an antibody or antigen-binding fragmentof an antibody that specifically binds PCSK9 and comprises the heavy andlight chain CDRs of a HCVR/LCVR amino acid sequence pair selected fromthe group consisting of SEQ ID Nos: 90/92 and 218/226.
 2. The method ofclaim 1, wherein the serum IDL-C is reduced in the patient by at leastabout 50% to about 56% from baseline following administration of thepharmaceutical composition.
 3. The method of claim 1, wherein thepharmaceutical composition comprises 20 mg to 200 mg of the PCSK9inhibitor.
 4. The method of claim 3, wherein the pharmaceuticalcomposition comprises 75 mg or 150 mg of the PCSK9 inhibitor.
 5. Themethod of claim 1, wherein the antibody or antigen-binding fragmentthereof comprises heavy and light chain CDR amino acid sequences havingSEQ ID NOs: 220, 222, 224, 228, 230 and
 232. 6. The method of claim 5,wherein the antibody or antigen-binding fragment thereof comprises anHCVR having the amino acid sequence of SEQ ID NO:218 and an LCVR havingthe amino acid sequence of SEQ ID NO:226.
 7. The method of claim 1,wherein the antibody or antigen-binding fragment thereof comprises heavyand light chain CDR amino acid sequences having SEQ ID NOs: 76, 78, 80,84, 86 and
 88. 8. The method of claim 7, wherein the antibody orantigen-binding fragment thereof comprises an HCVR having the amino acidsequence of SEQ ID NO:90 and an LCVR having the amino acid sequence ofSEQ ID NO:92.
 9. The method of claim 1, wherein the patient is on atherapeutic statin regimen at the time of or just prior toadministration of the pharmaceutical composition.
 10. The method ofclaim 9, wherein the therapeutic statin regimen comprises a statinselected from the group consisting of cerivastatin, atorvastatin,simvastatin, pitavastatin, rosuvastatin, fluvastatin, lovastatin andpravastatin.
 11. The method of claim 10, wherein the statin isatorvastatin.
 12. The method of claim 1, wherein the patient is not on atherapeutic statin regimen at the time of administration of thepharmaceutical composition.